Isolation and Differentiation of Naive CD4 T Cells
Naive CD4 T cells were isolated by FACS sorting using a MoFlo sorter of lymph nodes cell suspensions for CD44lo CD25− CD4+ cells. The culture mediums used were IMDM (Sigma-Aldrich) or RPMI 1640 (Sigma-Aldrich), both supplemented with 2 × 10−3 M l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 × 10−5 M β-mercaptoethanol, and 5% FCS. In some cases, RPMI medium was supplemented with 11 mg/liter l-tryptophan (Invitrogen) to adjust it to the concentrations found in IMDM. Th17 cells were differentiated on plates coated with 2 μg/ml anti-CD3 + 5 μg/ml anti-CD28 with a cytokine cocktail of 50 ng/ml IL-6, 1 ng/ml TGF-β, and 10 ng/ml IL-1. Neutralizing antibodies to IFN-γ, IL-4, or IL-2 were added at 10 μg/ml in some experiments. Th1 cells were stimulated in the presence of 3 ng/ml IL-12, and iT reg cells were generated by adding 10 ng/ml TGF-β. The AhR antagonist CH-223191 (Calbiochem) was added at 3 μM at the start of culture. FICZ was added at 300 nM at the start of some cultures. For measurements of intracellular cytokines, T cells were restimulated with 500 ng/ml phorbol dibutyrate and 500 ng/ml ionomycin in the presence of brefeldin A for 4 h on day 5 after initiation of cultures. Measurement of Stat5 phosphorylation was done with antibodies to pStat5 (BD) according to the manufacturer's instructions.
Partial Protocol Preview
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Veldhoen M., Hirota K., Christensen J., O'Garra A, & Stockinger B. (2009). Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells. The Journal of Experimental Medicine, 206(1), 43-49.
FRET-based Enzymatic Inhibition Assay for SARS-CoV-2 3CL Protease
The FRET-based enzymatic inhibition assay was utilized to measure the inhibitory activities of compounds against 3CL proteases. Primary screening was carried out to identify the inhibitors against SARS-CoV-2 3CLpro using an FDA-approved drug library containing an array of 1,018 compounds obtained from Selleck Chemicals (#L1300) [30] (link). The fluorogenic substrate Dabcyl-KNSTLQSGLRKE-Edans (GenScript, Shanghai, China) was synthesized to measure the protease activity of SARS-CoV-2 3CLpro or SARS-CoV 3CLpro according to our previous method [30] (link), [37] (link). Dabcyl-KTSAVLQSGFRKME-Edans (GenScript, Shanghai, China) was used for enzymatic inhibition assay of MERS-CoV 3CLpro and HCoV-229E 3CLpro as described previously [38] (link), [39] (link). The enzymatic inhibition assays of different proteases were performed as follows. 120 nM of SARS-CoV-2 3CLpro in the reaction buffer (0.1 M PBS, 1 mM EDTA, pH 7.4) incubated with varying concentrations of tested compounds for 1 h at 37 °C and then the substrate (final concentration 20 μM) was added to start the reaction. Fluorescence intensity was monitored continuously every 2 min for up to 20 min with an excitation wavelength of 340 nm and emission wavelength of 490 nm by using Spectramax® ID3 plate reader (Molecular Devices, California, USA). For the SARS-CoV 3CLpro inhibition assay, 860 nM protease was mixed with 40 μM substrate in assay buffer. To determine the effect of DTT or GSH on the enzymatic inhibition of SARS-CoV-2 3CLpro by Thonzonium bromide, we performed the enzymatic inhibition assay according to the literature [40] (link). SARS-CoV-2 3CLpro was preincubated in reaction buffer with Thonzonium bromide in the presence of 4 mM DTT or 1 mM GSH compared with in the absence of DTT or GSH buffer. In the MERS-CoV 3CLpro and HCoV-229E 3CLpro enzymatic inhibition assay, the concentration of the proteases was 2 and 1 μM, corresponding to the final substrate concentration of 40 and 20 μM, respectively. The IC50 values were calculated by fitting the curve of normalized inhibition ratio with the different concentration of test compounds.
Wang R., Zhai G., Zhu G., Wang M., Gong X., Zhang W., Ge G., Chen H, & Chen L. (2022). Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses. Bioorganic Chemistry, 130, 106264.
For assessing the ability to inhibit the main protease (3CLpro), the IC50 was the half-maximal inhibitory concentration of the substance at which the fluorescence level reduced by 50% compared to the value obtained without adding the inhibitor [9 (link),10 (link),34 (link)]. Fluorescence occurred due to cleavage of the peptide substrate DabcylKTSAVLQ↓SGFRKME(Edans)NH2 by 3CLpro protease. ML188 inhibitor ((R)-N-(4-(tert-Butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Ambeed Inc, USA) was used as a positive control. In the study, the signal was recorded using the CLARIOstar Plus instrument (BMG Labtech) at 355 and 460 nm for excitation and radiation, respectively, in the kinetic scan mode. Reaction mixtures containing TrisHCl buffer, fluorogenic substrate, 3CLpro, and the compound being tested were prepared and incubated for 30 min in a 384-well plate at 30 °C. The measurement for each compound was carried out in triplicate. The instrument was calibrated using a solution of the peptide that had undergone complete hydrolysis. The accompanying MARS Data Analysis software was used to calculate IC50 values.
Baev D.S., Blokhin M.E., Chirkova V.Y., Belenkaya S.V., Luzina O.A., Yarovaya O.I., Salakhutdinov N.F, & Shcherbakov D.N. (2022). Triterpenic Acid Amides as Potential Inhibitors of the SARS-CoV-2 Main Protease. Molecules, 28(1), 303.
To assess the ability to inhibit the main protease (3CLpro), the IC50 is the semi-inhibitory concentration of the substance, at which the fluorescence level is reduced by 50% relative to the value obtained without adding the inhibitor [30 (link)]. Fluorescence occurs due to cleavage of the peptide substrate DabcylKTSAVLQ↓SGFRKME(Edans)NH2 by 3CLpro protease. In the study, the signal was recorded using the CLARIOstar Plus instrument (BMG Labtech) at 355 and 460 nm for excitation/radiation, respectively, in kinetic scan mode. Reaction mixtures containing TrisHCl buffer, fluorogenic substrate, 3CLpro, and the compound being tested were prepared and incubated for 30 min in a 384-well plate at 30 °C. The instrument was calibrated using a solution of the peptide that had undergone complete hydrolysis. The accompanying MARS Data Analysis software (https://www.selectscience.net/products/mars-data-analysis-software/?prodID=81306, accessed on 18 August 2022) was used to calculate IC50.
Filimonov A.S., Yarovaya O.I., Zaykovskaya A.V., Rudometova N.B., Shcherbakov D.N., Chirkova V.Y., Baev D.S., Borisevich S.S., Luzina O.A., Pyankov O.V., Maksyutov R.A, & Salakhutdinov N.F. (2022). (+)-Usnic Acid and Its Derivatives as Inhibitors of a Wide Spectrum of SARS-CoV-2 Viruses. Viruses, 14(10), 2154.
The first step involved the curation and preparation of the experimental screening data. We downloaded the data on SARS-CoV-2 cytopathic effect (CPE) from the NCATS COVID-19 portal. This assay measures the ability of a compound to reverse the cytopathic effect induced by the virus in Vero E6 host cells (one of the most commonly used cell lines for studying this family of virus). The assay provides valuable basic information about the ability of a compound to restore the cells’ function but does not provide indication of the possible mechanism of action(s) of the small chemical compound under study. We also explored using the same Vero E6 cells the cytotoxic effect of these molecules in a counterscreen since the observed effect of some compounds may be mediated through cell viability and could lead to erroneous interpretation of the CPE results. Such information on cell viability is evidently important in the applicability of such compounds in clinical pharmacological interventions. Some additional data are important to take into account in the present analysis and involve the effect of the chemical compounds on human fibroblasts. This assay measures the host cell ATP content as a readout for cytotoxicity. When available, we annotated further the CPE data with the results of this assay. To be able to quickly repurpose a compound, we focused essentially on molecules that have been approved or are in clinical trials (in some instances, drugs are only approved for veterinary use). We thus cross-linked the CPE sample ID numbers to drug names (eg, international nonproprietary names), PubChem SID or CID numbers23 (link) or to ChEMBL ID numbers24 (link) and to molecules that possess ATC (Anatomical Therapeutic Chemical Classification system) codes and/or have documented therapeutic indications as found in DrugBank,25 (link) DrugCentral26 (link) and eDrug3d.27 (link) Molecules that are at the preclinical stage (eg, chemical probes) were essentially excluded via this procedure. Additionally, the salts, when present in the chemical file, were deleted using the Maya chemistry toolkit,28 (link) and the chemistry of the molecules was standardized using our FAF-Drugs4 web server.29 (link) The drugs in duplicate were removed. Further curation of the CPE data also involved the following: i) removal of a few molecules without potency values (AC50 values are reported for this dataset, ie, concentration for half-maximal activity30 (link)), ii) investigation of the curve class (ie, the evaluation of the quality of the dose–response curve and thus the quality of the measurements) in the CPE data (a curve class of type 4 is a flat curve indicating no activity) and maximum response (ie, maximum response value detected in the experiment). In such assay, compounds are usually considered active when the maximum response (absolute) value is above 30.31 Further, we removed molecules that were found highly cytotoxic in the Vero E6 counterscreen assay. From the initial CPE data containing 10,721 assessed samples (some molecules have been screened several times and most are chemical probes), we ended up with a list of 198 bioactive drugs. In this list of drugs, 93 were not tested for toxicity on human fibroblast and for the remaining 105 molecules, only one was found cytotoxic (Disulfiram, used for alcohol addiction, this molecule was also removed not only because of its cytotoxic nature in these assays but also as found to be a promiscuous SARS-CoV–2 main protease inhibitor32 (link)). At this stage of the analysis, we decided to retain all the compounds including those not annotated for human fibroblast toxicity. We then focused our attention on families of molecules that contain several members and removed some molecules with highly specific disease indication such as Deferasirox (an oral iron chelator given to patients receiving long-term blood transfusions). In this last step of data curation, we grouped the 198 compounds according to chemistry and disease indications and removed molecules that did not fulfill our selection criteria. This step led to the selection of a final list of 161 molecules reported in Table 1. Numerous approaches can be used to investigate low molecular weight chemical compounds in silico (eg,33–35 (link)) and here we decided to analyze these molecules with DataWarrior.36 (link) In this final list, very few molecules are only chemical probes, like Calpeptin (moderate activity against SARS-CoV-2 main protease Mpro). They were kept because they are often used to assess some enzymatic activities. The structural classification of chemical entities was then performed using the ClassyFire application.37 (link)
Investigated Chemical Probes and Approved Drugs
Name
Indication
AC50 (uM)
Fibroblast-Toxicity
ClinicalTrials.gov (Feb 24, 2021)
cLogP
Simple Rule-Based Estimation of Basic Nitrogen Atoms
Abemaciclib
Cancer
1.412537545
Not determined (ND)
No
4.1154
4
Acetopromazine
Psychotropic
12.58925412
No (N)
No
3.8721
1
Acolbifene
Cancer
11.22018454
ND
No
4.9846
1
Amiodarone
Cardiovascular
8.912509381
N
Yes
6.2801
1
Amitriptyline
Psychotropic
12.58925412
N
No
4.4056
1
Amlodipine
Cardiovascular
12.58925412
ND
Yes
2.071
1
Amodiaquine
Infectious
1.584893193
ND
Yes
4.2018
2
Amoxapine
Psychotropic
8.912509381
N
No
3.3035
2
Andrographolide
Infectious
11.22018454
ND
No
1.883
0
Anidulafungin
Infectious
5.623413252
ND
No
−0.4951
0
Apilimod
Infectious
12.58925412
ND
Yes
5.1942
2
Aprindine
Cardiovascular
8.912509381
N
No
4.5009
1
Arbidol (or Umifenovir)
Infectious
8.912509381
ND
Yes
4.1689
1
Aripiprazole
Psychotropic
12.58925412
N
No
4.4351
1
Asenapine
Psychotropic
10
ND
No
4.9405
1
Ataciguat (experimental)
Cardiovascular
12.58925412
ND
No
1.8036
0
Azelastine
Antihistamine
11.22018454
N
No
4.3744
1
Azithromycin
Infectious
11.22018454
N
Yes
1.6569
2
Bazedoxifene
Bone problems and possibly Cancer
3.981071706
N
No
5.6968
1
Benazepril
Cardiovascular
10
N
Yes
0.8715
1
Bencyclane
Cardiovascular
12.58925412
N
No
3.8639
1
Bepridil
Cardiovascular
8.912509381
N
No
4.4044
1
Berbamine (experimental)
Cancer
1.412537545
ND
No
6.2253
2
Berzosertib (experimental)
Cancer
2.238721139
ND
No
1.669
1
Bexarotene
Cancer
12.58925412
N
No
4.799
0
Bifemelane (approved Japan)
Psychotropic
12.58925412
N
No
3.7476
1
Blonanserin (Japan)
Psychotropic
5.623413252
N
No
5.3682
2
Brexpiprazole
Psychotropic
11.22018454
ND
No
3.8983
1
Bromodiphenhydramine
Antihistamine
12.58925412
N
No
3.631
1
Buclizine
Antihistamine
12.58925412
N
No
6.6827
2
Calpeptin (experimental)
Cancer
0.316227766
ND
No
2.8358
0
Carvedilol
Cardiovascular
11.22018454
ND
Yes
3.1668
1
Cenicriviroc
Infectious
8.912509381
ND
Yes
7.2606
1
Ceritinib
Cancer
10
ND
No
5.5817
3
Chlorcyclizine
Antihistamine
12.58925412
N
No
3.6827
2
Chloroquine
Infectious
3.981071706
N
Yes
4.0091
2
Chloroxine
Infectious
12.58925412
N
No
2.8419
0
Chlorpromazine
Psychotropic
8.912509381
N
Yes
4.6069
1
Chlorprothixene
Psychotropic
3.548133892
N
No
5.1216
1
Ciclopirox
Infectious
5.011872336
N
No
1.4285
0
Cilnidipine
Cardiovascular
11.22018454
N
No
3.6409
0
Clemastine
Antihistamine
11.22018454
N
No
4.5988
1
Clioquinol (withdrawn)
Infectious
10
N
No
2.673
0
Clofazimine
Infectious
6.309573445
ND
Yes
6.2781
1
Clomipramine
Psychotropic
7.943282347
N
No
4.4969
1
Closantel (veterinary drug, likely toxic in humans)
Infectious
11.22018454
N
No
6.4242
0
Clozapine
Psychotropic
12.58925412
N
No
3.2447
2
Cyclobenzaprine
Initially used as Psychotropic
10
N
No
4.3534
1
Cyproheptadine
Antihistamine
3.981071706
N
No
4.5999
1
Cysmethynil (experimental)
Cancer
10
ND
No
5.9027
0
Dabrafenib
Cancer
12.58925412
ND
No
3.666
0
Danazol
Cancer and endometriosis
12.58925412
N
No
3.4638
0
Dapoxetine
Initially psychotropic agent but now premature ejaculation
12.58925412
N
No
4.2367
1
Deserpidine
Cardiovascular
12.58925412
ND
No
3.6454
1
Desipramine
Psychotropic
8.912509381
N
No
3.6244
1
Desloratadine
Antihistamine
11.22018454
N
No
4.0583
1
Desmethylclozapine (metabolite of clozapine)
Psychotropic
8.912509381
ND
No
2.9918
2
Dexanabinol (experimental)
Cancer
10
ND
No
6.9355
0
Difeterol
Antihistamine
12.58925412
N
No
4.0432
1
Doramectin (veterinary drug)
Infectious
12.58925412
ND
No
5.3516
0
Dosulepin
Psychotropic
12.58925412
ND
No
3.8201
1
Doxazosin
Cancer and hypertension
12.58925412
N
Yes
2.1379
2
Duloxetine
Psychotropic
8.912509381
N
No
3.8368
1
Efavirenz
Infectious
10
N
No
3.6614
0
Emodepside (veterinary drug)
Infectious
7.079457844
ND
No
5.237
0
Enzastaurin (experimental)
Cancer
12.58925412
N
No
3.1099
1
Flumatinib (experimental)
Cancer
7.943282347
ND
No
3.8397
2
Flunarizine
Antihistamine
11.22018454
N
No
5.4576
2
Fluoxetine
Psychotropic
4.466835922
N
Yes
3.6241
1
Fluphenazine-decanoate
Psychotropic
12.58925412
N
No
8.527
2
Fonazine (Japan and Europe)
Antihistamine
28.18382931
ND
No
3.1244
1
Halofantrine
Infectious
11.22018454
N
No
8.1762
1
Haloperidol
Psychotropic
25.11886432
N
No
4.3049
1
Hesperadin (experimental)
Cancer
12.58925412
ND
No
2.3768
1
Hexachlorophene
Infectious
0.223872114
N
No
6.4201
0
Hexetidine
Infectious
3.548133892
N
No
5.2587
1
Homochlorcyclizine (Japan)
Antihistamine
5.011872336
N
No
4.0247
2
Hycanthone
Infectious
12.58925412
ND
No
3.3212
1
Hydroquinidine
Cardiovascular
12.58925412
ND
No
2.7959
1
Icaritin
Cancer
12.58925412
ND
No
4.1341
0
Iloperidone
Psychotropic
12.58925412
N
No
4.3044
1
Imatinib
Cancer
5.623413252
N
Yes
3.9383
2
Imipramine
Psychotropic
8.912509381
N
No
3.8909
1
Lapatinib
Cancer
10
N
No
4.7281
1
Lemildipine (experimental)
Cardiovascular
12.58925412
N
No
3.3887
0
Lercanidipine
Cardiovascular
11.22018454
N
No
4.7102
1
Letermovir
Infectious
10
ND
No
4.8615
1
Lomerizine
Antihistamine (treatment of migraines)
12.58925412
N
No
4.4864
2
Lopinavir
Infectious
12.58925412
N
Yes
4.847
0
Loratadine
Antihistamine
10
N
No
4.9774
0
Loxapine
Psychotropic
10
N
No
3.5564
1
Manassantin-A (experimental)
Cancer
3.16227766
ND
No
5.8318
0
Manidipine
Cardiovascular
8.912509381
N
No
2.6132
2
Maprotiline
Psychotropic
8.912509381
N
No
3.7029
1
Masitinib
Cancer
11.22018454
ND
Yes
4.7573
3
Mefloquine
Infectious
11.22018454
ND
Yes
3.5076
1
Melitracen
Psychotropic
11.22018454
ND
No
4.6647
1
Merimepodib
Infectious
12.58925412
ND
Yes
2.7196
0
Mesoridazine
Psychotropic
10
N
No
4.5579
1
Methdilazine
Antihistamine
7.079457844
ND
No
4.2005
1
Methotrimeprazine
Psychotropic
12.58925412
ND
No
4.1492
1
Momelotinib (experimental)
Cancer
12.58925412
ND
No
2.5062
0
Monatepil (experimental)
Cardiovascular
12.58925412
N
No
4.7269
1
Nafronyl
Psychotropic
12.58925412
ND
No
4.2981
1
Naftopidil
Cancer
12.58925412
N
No
3.42
1
Nicardipine
Cardiovascular
10
N
No
1.5582
1
Niraparib
Cancer
12.58925412
ND
No
2.0619
1
Nirogacestat
Cancer
12.58925412
ND
No
4.3631
3
Nitazoxanide
Infectious
3.16227766
N
Yes
1.6871
0
Nitroxoline
Infectious
12.58925412
ND
No
0.7083
0
Oxatomide
Antihistamine
10
N
No
4.2761
2
Pamelor
Psychotropic
12.58925412
ND
No
4.1391
1
Parthenolide
Cancer
10
ND
No
2.7525
0
Pazopanib
Cancer
12.58925412
ND
No
0.9125
2
Periciazine
Psychotropic
10
ND
No
4.1174
1
Perphenazine
Antihistamine and psychotropic
12.58925412
N
No
4.1649
2
Pipequaline (experimental)
Psychotropic
12.58925412
ND
No
4.9763
1
Piperacetazine (prodrug)
Psychotropic
12.58925412
N
No
4.8021
1
Piroctone (experimental)
Infectious and Cancer
7.943282347
ND
No
2.9044
0
Pizotifen
Psychotropic and antihistamine
10
N
No
4.5187
1
Pluripotin (experimental)
Cancer
12.58925412
ND
No
4.2874
0
Prochlorperazine
Psychotropic
8.912509381
N
No
4.6853
2
Promazine
Psychotropic
10
N
No
4.0009
1
Promethazine
Antihistamine
8.912509381
N
Yes
3.9058
1
Propafenone
Cardiovascular
12.58925412
N
No
3.0837
1
Propionylpromazine (veterinary drug)
Antihistamine and neuroleptic
10
N
No
4.3265
1
Protriptyline
Psychotropic
12.58925412
N
No
3.6848
1
Pyrimethamine
Infectious
4.466835922
ND
No
2.5414
2
Quinidine
Cardiovascular
12.58925412
ND
No
2.6104
1
Quizartinib (experimental)
Cancer
2.511886432
ND
No
4.7979
2
Refametinib (experimental)
Cancer
11.22018454
ND
No
1.88
0
Remdesivir
Infectious
7.943282347
ND
Yes
0.3048
0
Rescimetol (Japan)
Cardiovascular
12.58925412
N
No
3.6989
1
Reserpine
Cardiovascular
11.22018454
N
Yes
3.5754
1
Retapamulin
Infectious
12.58925412
ND
No
5.218
1
Sarpogrelate
Cardiovascular
12.58925412
N
No
1.8567
1
Serdemetan (experimental)
Cancer
8.912509381
ND
No
3.7237
1
Siccanin (experimental)
Infectious
12.58925412
ND
No
4.3637
0
Spiclomazine (experimental)
Cancer
12.58925412
ND
No
5.2169
1
Spiperone (Japan)
Psychotropic
7.943282347
N
No
3.0219
1
Spiramycin-II
Infectious
12.58925412
N
No
2.4608
2
Sulfatinib (experimental)
Cancer
12.58925412
ND
No
2.0918
1
Teicoplanin
Infectious
14.12537545
ND
Yes
−3.4194
1
Thioproperazine
Psychotropic
11.22018454
N
No
3.2979
2
Thiothixene
Psychotropic
12.58925412
ND
No
2.937
2
Tilorone
Infectious
10
ND
No
4.0607
2
Timiperone (Japan)
Psychotropic
10
ND
No
3.7407
1
Tioguanine
Cancer
10
ND
No
−0.9683
1
Tipifarnib (experimental)
Cancer
12.58925412
ND
No
4.03
2
Tivantinib (experimental)
Cancer
12.58925412
ND
No
4.1837
0
Tizoxanide
Infectious
3.16227766
ND
Yes
1.3547
0
Triamterene
Cardiovascular
7.079457844
N
Yes
0.6075
2
Trifluomeprazine
Psychotropic
10
ND
No
5.0675
1
Trifluoperazine
Psychotropic
12.58925412
ND
No
4.9276
2
Triflupromazine
Psychotropic
11.22018454
N
No
4.8492
1
Trimeprazine (or Alimemazine)
Psychotropic
8.912509381
N
No
4.2192
1
Trimetrexate
Cancer
10
ND
No
1.9786
2
Trimipramine
Psychotropic
10
N
No
4.1092
1
Vilazodone
Psychotropic
10
ND
No
3.6629
1
Vorapaxar
Cardiovascular
8.912509381
ND
No
4.7865
0
Zotepine
Psychotropic
12.58925412
N
No
4.6741
1
Notes: The names of the 161 selected bioactive molecules are reported in the first column. For each compound, the main indication, the experimental AC50 values (see text), the experimental fibroblast toxicity (ND, not determined, N, No), the computed log P (cLogP), and a simple estimation of the number of basic nitrogen atoms are shown. Also, molecules that are in clinical trials for COVID-19 treatments indicated; Experimental and investigational drugs are all labelled in this Table experimental.
Villoutreix B.O., Krishnamoorthy R., Tamouza R., Leboyer M, & Beaune P. (2021). Chemoinformatic Analysis of Psychotropic and Antihistaminic Drugs in the Light of Experimental Anti-SARS-CoV-2 Activities. Advances and Applications in Bioinformatics and Chemistry : AABC, 14, 71-85.
Fluorescence Polarization Assay for Mpro Inhibitor Screening
In this FP screening assay, 29 µL sample of 400 nM Mpro diluted in the FP assay buffer was mixed with 1 µL of natural product (1 mg/mL in DMSO) in a black 384-well microplate, and the mixture was further incubated for 35 min at RT before adding 20 µL sample of 60 nM FP tracer. After proceeding for 20 min at RT, the reaction was quenched by addition of 10 µL sample of 300 nM avidin, and the mP values were measured by a microplate reader. In each assay plate, GC-376 (1 µM) and DMSO were used as a positive and negative control, respectively. The well containing only 60 µL sample of 20 nM FITC-AVLQ peptide was used to assess the background noise. The inhibitory activity of screening compound was calculated using Eq. (1), and the candidate compounds (> 50% inhibition) were analyzed in triplicate to generate the dose-response curves in the second screening. The inhibitory activity of dieckol in this FP screening assay was carried out as mentioned above. The initial concentration of dieckol was 100 µM, and 8 two-fold dilutions were prepared for the determination of IC50 value. To remove the DTT effect on the enzymatic assay [26 (link)], the IC50 value was also obtained in the absence of DTT in a FP screening assay.
Yan G., Li D., Lin Y., Fu Z., Qi H., Liu X., Zhang J., Si S, & Chen Y. (2021). Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors. Cell & Bioscience, 11, 199.
Supplemented with L-glutamine, penicillin, streptomycin, β-mercaptoethanol, and FCS
RPMI medium supplemented with L-tryptophan
Phorbol dibutyrate and ionomycin stimulation for intracellular cytokine measurement
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